Phage display has been intensively used for producing combinatorial antibody libraries and for presentation of combinatorial arrays of peptide elements (see, e.g., Rodi et al, Curr. Opin. Biotechnol., 10:87-93, 1999; Vaughan et al, Nat. Biotechnol., 16:535-539, 1998; Griffiths et al, Curr. Opin. Biotechnol., 9:102-108, 1998; Zwick et al, Curr. Opin. Biotechnol., 9:427-436, 1998; Dall'Acqua et al, Curr. Opin. Struct. Biol., 8:443-450, 1998; Raag et al, Faseb J., 9:73-80, 1995; Barbas et al, Proc. Natl. Acad. Sci. USA, 88:7978-7982, 1991; Kang et al, Proc. Natl. Acad. Sci. USA, 88:4363-4366, 1991; Huse et al, Science, 246:1273-1278, 1989; each herein incorporated by reference in its entirety).
However, many details of the phage particle itself have not been fully elucidated and the possibility of alternative display formats also remains to be explored. The filamentous bacteriophage fd, and similarly M13, consists of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins (FIG. 1). The molecular mass of a particle is about 1.6×107 Da, of which 88% is comprised of protein and 12% is comprised of DNA (Berkowitz et al, J. Mol. Biol., 102:531-547, 1976; herein incorporated by reference in its entirety). Approximately 2700 molecules of the major coat protein pVIII surround each phage particle. At one end of the particle, there are five copies each of pIII and pVI that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are hydrophobic peptides of 33 and 32 amino acids, respectively, required for the initiation of assembly and for maintenance of virion stability. While pIII, pVI, and pVIII have been used to display biological molecules, pVII and pIX have historically been less often utilized (Rodi et al, Curr. Opin. Biotechnol., 10:87-93, 1999; Russel et al, J. Virol., 63:3284-3295, 1989; U.S. patent application Ser. No. 10/222,026; each herein incorporated by reference in its entirety).
Initially it had been found that attempts at phage assembly in the absence of pIX almost completely abolish the production of phage. In addition, initial attempts at displaying a fusion protein on pIX suggested that pIX was not functional when fused with another protein at its N-terminus (Endemann et al, J. Mol. Biol., 250:496-506, 1995), suggesting that display would not be feasible using pIX. However, subsequent work has proven the feasibility of using pIX for fusion proteins in phage display (e.g., U.S. patent application Ser. No. 10/222,026 and WO 2010/097411; herein incorporated by reference in their entirety).
However, prior to development of some embodiments of the present invention, application of pIX fusion proteins remained limited to mono- or oligovalent display of protein-of-interest:pIX fusions. Multivalent display is of benefit in certain applications, including but not limited to applications where a reduction in affinity of the retrieved entities is desired (such as when it is desirable to isolate rare and/or weakly binding clones during de novo selection of peptides binding a particular target), or when conducting stability engineering strategies.
Therefore, what is needed is improved methods, systems, and compositions for multivalent phagemid-based phage display.